Pattern formation is the activity by which embryonic cells form ordered spatial arrangements of differentiated tissues. The physical complexity of higher organisms arises during embryogenesis through the interplay of cell-intrinsic lineage and cell-extrinsic signaling. Inductive interactions are essential to embryonic patterning in vertebrate development from the earliest establishment of the body plan, to the patterning of the organ systems, to the generation of diverse cell types during tissue differentiation (Davidson, E., (1990) Development 108: 365-389; Gurdon, J. B., (1992) Cell 68: 185-199; Jessell, T. M. et al., (1992) Cell 68: 257-270). The effects of developmental cell interactions are varied. Typically, responding cells are diverted from one route of cell differentiation to another by inducing cells that differ from both the uninduced and induced states of the responding cells (inductions). Sometimes cells induce their neighbors to differentiate like themselves (homeogenetic induction); in other cases a cell inhibits its neighbors from differentiating like itself. Cell interactions in early development may be sequential, such that an initial induction between two cell types leads to a progressive amplification of diversity. Moreover, inductive interactions occur not only in embryos, but in adult cells as well, and can act to establish and maintain morphogenetic patterns as well as induce differentiation (J. B. Gurdon (1992) Cell 68:185-199).
Members of the Hedgehog family of signaling molecules mediate many important short- and long-range patterning processes during invertebrate and vertebrate development. In the fly, a single hedgehog gene regulates segmental and imaginal disc patterning. In contrast, in vertebrates, a hedgehog gene family is involved in the control of left-right asymmetry, polarity in the CNS, somites and limb, organogenesis, chondrogenesis and spermatogenesis.
The first hedgehog gene was identified by a genetic screen in the fruitfly Drosophila melanogaster (Nxc3xcsslein-Volhard, C. and Wieschaus, E. (1980) Nature 287, 795-801). This screen identified a number of mutations affecting embryonic and larval development. In 1992 and 1993, the molecular nature of the Drosophila hedgehog (hh) gene was reported (C. F., Lee et al. (1992) Cell 71, 33-50), and since then, several hedgehog homologues have been isolated from various vertebrate species. While only one hedgehog gene has been found in Drosophila and other invertebrates, multiple Hedgehog genes are present in vertebrates.
The vertebrate family of hedgehog genes includes at least four members, e.g., paralogs of the single drosophila hedgehog gene. Exemplary hedgehog genes and proteins are described in PCT publications WO 95/18856 and WO 96/17924. Three of these members, herein referred to as Desert hedgehog (Dhh), Sonic hedgehog (Shh) and Indian hedgehog (Ihh), apparently exist in all vertebrates, including fish, birds, and mammals. A fourth member, herein referred to as tiggie-winkle hedgehog (Thh), appears specific to fish. Desert hedgehog (Dhh) is expressed principally in the testes, both in mouse embryonic development and in the adult rodent and human; Indian hedgehog (Ihh) is involved in bone development during embryogenesis and in bone formation in the adult; and, Shh, which as described above, is primarily involved in morphogenic and neuroinductive activities. Given the critical inductive roles of hedgehog polypeptides in the development and maintenance of vertebrate organs, the identification of hedghog interacting proteins is of paramount significance in both clinical and research contexts.
The various Hedgehog proteins consist of a signal peptide, a highly conserved N-terminal region, and a more divergent C-terminal domain. In addition to signal sequence cleavage in the secretory pathway (Lee, J. J. et al. (1992) Cell 71:33-50; Tabata, T. et al. (1992) Genes Dev. 2635-2645; Chang, D. E. et al. (1994) Development 120:3339-3353), Hedgehog precursor proteins undergo an internal autoproteolytic cleavage which depends on conserved sequences in the C-terminal portion (Lee et al. (1994) Science 266:1528-1537; Porter et al. (1995) Nature 374:363-366). This autocleavage leads to a 19 kD N-terminal peptide and a C-terminal peptide of 26-28 kD (Lee et al. (1992) supra; Tabata et al. (1992) supra; Chang et al. (1994) supra; Lee et al. (1994) supra; Bumcrot, D. A., et al. (1995) Mol. Cell. Biol. 15:2294-2303; Porter et al. (1995) supra; Ekker, S. C. et al. (1995) Curr. Biol. 5:944-955; Lai, C. J. et al. (1995) Development 121:2349-2360). The N-terminal peptide stays tightly associated with the surface of cells in which it was synthesized, while the C-terminal peptide is freely diffusible both in vitro and in vivo (Porter et al. (1995) Nature 374:363; Lee et al. (1994) supra; Bumcrot et al. (1995) supra; Mart"", E. et al. (1995) Development 121:2537-2547; Roelink, H. et al. (1995) Cell 81:445-455). Interestingly, cell surface retention of the N-terminal peptide is dependent on autocleavage, as a truncated form of HH encoded by an RNA which terminates precisely at the normal position of internal cleavage is diffusible in vitro (Porter et al. (1995) supra) and in vivo (Porter, J. A. et al. (1996) Cell 86, 21-34). Biochemical studies have shown that the autoproteolytic cleavage of the HH precursor protein proceeds through an internal thioester intermediate which subsequently is cleaved in a nucleophilic substitution. It is likely that the nucleophile is a small lipophilic molecule which becomes covalently bound to the C-terminal end of the N-peptide (Porter et al. (1996) supra), tethering it to the cell surface. The biological implications are profound. As a result of the tethering, a high local concentration of N-terminal Hedgehog peptide is generated on the surface of the Hedgehog producing cells. It is this N-terminal peptide which is both necessary and sufficient for short- and long-range Hedgehog signaling activities in Drosophila and vertebrates (Porter et al. (1995) supra; Ekker et al. (1995) supra: Lai et al. (1995) supra; Roelink, H. et al. (1995) Cell 81:445-455; Porter et al. (1996) supra; Fietz, M. J. et al. (1995) Curr. Biol. 5:643-651; Fan, C. -M. et al. (1995) Cell 81:457-465; Mart"", E., et al. (1995) Nature 375:322-325; Lopez-Martinez et al. (1995) Curr. Biol 5:791-795; Ekker, S. C. et al. (1995) Development 121:2337-2347; Forbes, A. J. et al.(1996) Development 122:1125-1135).
HH has been implicated in short- and long-range patterning processes at various sites during Drosophila development. In the establishment of segment polarity in early embryos, it has short-range effects which appear to be directly mediated, while in the patterning of the imaginal discs, it induces long range effects via the induction of secondary signals.
In vertebrates, several hedgehog genes have been cloned in the past few years. Of these genes, Shh has received most of the experimental attention, as it is expressed in different organizing centers which are the sources of signals that pattern neighboring tissues. Recent evidence indicates that Shh is involved in these interactions.
The expression of Shh starts shortly after the onset of gastrulation in the presumptive midline mesoderm, the node in the mouse (Chang et al. (1994) supra; Echelard, Y. et al. (1993) Cell 75:1417-1430), the rat (Roelink, H. et al. (1994) Cell 76:761-775) and the chick (Riddle, R. D. et al. (1993) Cell 75:1401-1416), and the shield in the zebrafish (Ekker et al. (1995) supra; Krauss, S. et al.(1993) Cell 75:1431-1444). In chick embyros, the Shh expression pattern in the node develops a left-right asymmetry, which appears to be responsible for the left-right situs of the heart (Levin, M. et al. (1995) Cell 82:803-814).
In the CNS, Shh from the notochord and the floorplate appears to induce ventral cell fates. When ectopically expressed, Shh leads to a ventralization of large regions of the mid- and hindbrain in mouse (Echelard et al. (1993) supra; Goodrich, L. V. et al. (1996) Genes Dev. 10:301-312), Xenopus (Roelink, H. et al. (1994) supra; Ruiz i Altaba, A. et al. (1995) Mol. Cell. Neurosci. 6:106-121), and zebrafish (Ekker et al. (1995) supra; Krauss et al. (1993) supra; Hammerschmidt, M., et al. (1996) Genes Dev. 10:647-658). In explants of intermediate neuroectoderm at spinal cord levels, Shh protein induces floorplate and motor neuron development with distinct concentration thresholds, floor plate at high and motor neurons at lower concentrations (Roelink et al. (1995) supra; Mart"" et al. (1995) supra; Tanabe, Y. et al. (1995) Curr. Biol. 5:651-658). Moreover, antibody blocking suggests that Shh produced by the notochord is required for notochord-mediated induction of motor neuron fates (Mart"" et al. (1995) supra). Thus, high concentration of Shh on the surface of Shh-producing midline cells appears to account for the contact-mediated induction of floorplate observed in vitro (Placzek, M. et al. (1993) Development 117:205-218), and the midline positioning of the floorplate immediately above the notochord in vivo. Lower concentrations of Shh released from the notochord and the floorplate presumably induce motor neurons at more distant ventrolateral regions in a process that has been shown to be contact-independent in vitro (Yamada, T. et al. (1993) Cell 73:673-686). In explants taken at midbrain and forebrain levels, Shh also induces the appropriate ventrolateral neuronal cell types, dopaminergic (Heynes, M. et al. (1995) Neuron 15:35-44; Wang, M. Z. et al. (1995) Nature Med. 1:1184-1188) and cholinergic (Ericson, J. et al. (1995) Cell 81:747-756) precursors, respectively, indicating that Shh is a common inducer of ventral specification over the entire length of the CNS. These observations raise a question as to how the differential response to Shh is regulated at particular anteroposterior positions.
Shh from the midline also patterns the paraxial regions of the vertebrate embryo, the somites in the trunk (Fan et al. (1995) supra) and the head mesenchyme rostral of the somites (Hammerschmidt et al. (1996) supra). In chick and mouse paraxial mesoderm explants, Shh promotes the expression of sclerotome specific markers like Pax1 and Twist, at the expense of the dermamyotomal marker Pax3. Moreover, filter barrier experiments suggest that Shh mediates the induction of the sclerotome directly rather than by activation of a secondary signaling mechanism (Fan, C. -M. and Tessier-Lavigne, M. (1994) Cell 79, 1175-1186).
Shh also induces myotomal gene expression (Hammerschmidt et al. (1996) supra; Johnson, R. L. et al. (1994) Cell 79:1165-1173; Mxc3xcnsterberg, A. E. et al. (1995) Genes Dev. 9:2911-2922; Weinberg, E. S. et al. (1996) Development 122:271-280), although recent experiments indicate that members of the WNT family, vertebrate homologues of Drosophila wingless, are required in concert (Mxc3xcnsterberg et al. (1995) supra). Puzzlingly, myotomal induction in chicks requires higher Shh concentrations than the induction of sclerotomal markers (Mxc3xcnsterberg et al. (1995) supra), although the sclerotome originates from somitic cells positioned much closer to the notochord. Similar results were obtained in the zebrafish, where high concentrations of Hedgehog induce myotomal and repress sclerotomal marker gene expression (Hammerschmidt et al. (1996) supra). In contrast to amniotes, however, these observations are consistent with the architecture of the fish embryo, as here, the myotome is the predominant and more axial component of the somites. Thus, modulation of Shh signaling and the acquisition of new signaling factors may have modified the somite structure during vertebrate evolution.
In the vertebrate limb buds, a subset of posterior mesenchymal cells, the xe2x80x9cZone of polarizing activityxe2x80x9d (ZPA), regulates anteroposterior digit identity (reviewed in Honig, L. S. (1981) Nature 291:72-73). Ectopic expression of Shh or application of beads soaked in Shh peptide mimics the effect of anterior ZPA grafts, generating a mirror image duplication of digits (Chang et al. (1994) supra; Lopez-Martinez et al. (1995) supra; Riddle et al. (1993) supra) (FIG. 2g). Thus, digit identity appears to depend primarily on Shh concentration, although it is possible that other signals may relay this information over the substantial distances that appear to be required for AP patterning (100-150 xcexcm). Similar to the interaction of HH and DPP in the Drosophila imaginal discs, Shh in the vertebrate limb bud activates the expression of Bmp2 (Francis, P. H. et al. (1994) Development 120:209-218), a dpp homologue. However, unlike DPP in Drosophila, Bmp2 fails to mimic the polarizing effect of Shh upon ectopic application in the chick limb bud (Francis et al. (1994) supra). In addition to anteroposterior patterning, Shh also appears to be involved in the regulation of the proximodistal outgrowth of the limbs by inducing the synthesis of the fibroblast growth factor FGF4 in the posterior apical ectodermal ridge (Laufer, E. et al. (1994) Cell 79:993-1003; Niswander, L. et al.(1994) Nature 371:609-612).
The close relationship between Hedgehog proteins and BMPs is likely to have been conserved at many, but probably not all sites of vertebrate Hedgehog expression. For example, in the chick hindgut, Shh has been shown to induce the expression of Bmp4, another vertebrate dpp homologue (Roberts, D. J. et al. (1995) Development 121:3163-3174). Furthermore, Shh and Bmp2, 4, or 6 show a striking correlation in their expression in epithelial and mesenchymal cells of the stomach, the urogential system, the lung, the tooth buds and the hair follicles (Bitgood, M. J. and McMahon, A. P. (1995) Dev. Biol. 172:126-138). Further, Ihh, one of the two other mouse Hedgehog genes, is expressed adjacent to Bmp expressing cells in the gut and developing cartilage (Bitgood and McMahon (1995) supra).
Recent evidence suggests a model in which Indian hedgehog (Ihh) plays a crucial role in the regulation of chondrogenic development (Roberts et al. (1995) supra). During cartilage formation, chondrocytes proceed from a proliferating state via an intermediate, prehypertrophic state to differentiated hypertrophic chondrocytes. Ihh is expressed in the prehypertrophic chondrocytes and initiates a signaling cascade that leads to the blockage of chondrocyte differentiation. Its direct target is the perichondrium around the Ihh expression domain, which responds by the expression of Gli and Patched (Ptc), conserved transcriptional targets of Hedgehog signals (see below). Most likely, this leads to secondary signaling resulting in the synthesis of parathyroid hormone-related protein (PTHrP) in the periarticular perichondrium. PTHrP itself signals back to the prehypertrophic chondrocytes, blocking their further differentiation. At the same time, PTHrP represses expression of Ihh, thereby forming a negative feedback loop that modulates the rate of chondrocyte differentiation.
Patched was originally identified in Drosophila as a segment polarity gene, one of a group of developmental genes that affect cell differentiation within the individual segments that occur in a homologous series along the anterior-posterior axis of the embryo. See Hooper, J. E. et al. (1989) Cell 59:751; and Nakano, Y. et al. (1989) Nature 341:508. Patterns of expression of the vertebrate homologue of patched suggest its involvement in the development of neural tube, skeleton, limbs, craniofacial structure, and skin.
Genetic and functional studies demonstrate that patched is part of the hedgehog signaling cascade, an evolutionarily conserved pathway that regulates expression of a number of downstream genes. See Perrimon, N. (1995) Cell 80:517; and Perrimon, N. (1996) Cell 86:513. Patched participates in the constitutive transcriptional repression of the target genes; its effect is opposed by a secreted glycoprotein, encoded by hedgehog, or a vertebrate homologue, which induces transcriptional activation. Genes under control of this pathway include members of the Wnt and TGF-beta families.
Patched proteins possess two large extracellular domains, twelve transmembrane segments, and several cytoplasmic segments. See Hooper, supra; Nakano, supra; Johnson, R. L. et al. (1996) Science 272:1668; and Hahn, H. et al. (1996) Cell 85:841. The biochemical role of patched in the hedgehog signaling pathway is unclear. Direct interaction with the hedgehog protein has, however, been reported (Chen, Y. et al. (1996) Cell 87:553), and patched may participate in a hedgehog receptor complex along with another transmembrane protein encoded by the smoothened gene. See Perrimon, supra; and Chen, supra.
The human homologue of patched was recently cloned and mapped to chromosome 9q22.3. See Johnson, supra; and Hahn, supra. This region has been implicated in basal cell nevus syndrome (BCNS), which is characterized by developmental abnormalities including rib and craniofacial alterations, abnormalities of the hands and feet, and spina bifida.
BCNS also predisposes to multiple tumor types, the most frequent being basal cell carcinomas (BCC) that occur in many locations on the body and appear within the first two decades of life. Most cases of BCC, however, are unrelated to the syndrome and arise sporadically in small numbers on sun-exposed sites of middle-aged or older people of northern European ancestry.
Recent studies in BCNS-related and sporadic BCC suggest that a functional loss of both alleles of patched leads to development of BCC. See Johnson, supra; Hahn, supra; and Gailani, M. R. et al. (1996) Nature Genetics 14:78. Single allele deletions of chromosome 9q22.3 occur frequently in both sporadic and hereditary BCC. Linkage analysis revealed that the defective inherited allele was retained and the normal allele was lost in tumors from BCNS patients.
Sporadic tumors also demonstrated a loss of both functional alleles of patched. Of twelve tumors in which patched mutations were identified with a single strand conformational polymorphism screening assay, nine had chromosomal deletion of the second allele and the other three had inactivating mutations in both alleles (Gailani, supra). The alterations did not occur in the corresponding germline DNA.
Most of the identified mutations resulted in premature stop codons or frame shifts. Lench, N. J., et al., Hum. Genet. 1997 October; 100(5-6): 497-502. Several, however, were point mutations leading to amino acid substitutions in either extracellular or cytoplasmic domains. These sites of mutation may indicate functional importance for interaction with extracellular proteins or with cytoplasmic members of the downstream signaling pathway.
The involvement of patched in the inhibition of gene expression and the occurrence of frequent allelic deletions of patched in BCC support a tumor suppressor function for this gene. Its role in the regulation of gene families known to be involved in cell signaling and intercellular communication provides a possible mechanism of tumor suppression.
The present invention makes available methods and reagents for regulating aberrant activity of the hedgehog signaling pathway, such as hedgehog gain-of-function, ptc loss-of-function, smoothened gain-of-function, comprising contacting the cell with a ptc agonist, such as a steroidal alkaloid or other small molecule, in a sufficient amount to antagonize the hedgehog pathway, e.g., to agonize a normal ptc pathway or antagonize smoothened activity. The present invention also makes available methods and reagents for regulating aberrant activity of the hedgehog signaling pathway, such as hedgehog loss-of-function, ptc gain-of-function, smoothened loss-of-function, comprising contacting the cell with a ptc antagonist, such as a steroidal alkaloid or other small molecule, in a sufficient amount to antagonize the hedgehog pathway, e.g., to agonize a normal ptc pathway or antagonize smoothened activity.
Furthermore, in light of the discovery that increased levels of cyclic adenosine monophosphate (cAMP) deactivate the hedgehog signalling pathway to inhibit ptc loss-of-function, hedgehog gain-of-function, or smoothened gain-of-function, the present invention makes available methods and reagents which raise cAMP levels for inhibiting aberrant growth states resulting from activation of this pathway. Suitable compounds include compounds which interact with G-protein coupled receptors, adenylate cyclase agonists, cAMP analogs, and cAMP phosphodiesterase antagonists. The subject method comprises contacting the cell with one or more such agents, preferably small molecules, in an amount sufficient to reverse or control the aberrant growth state, e.g., to agonize a normal ptc pathway, antagonize a normal hedgehog pathway, or antagonize smoothened activity.
Alternatively, an agent which promotes decreased levels of cAMP may be employed to inhibit ptc gain-of-function, hedgehog loss-of-function, or smoothened loss-of-function may be used in methods and reagents for inhibiting aberrant growth states resulting from deactivation of the hedgehog pathway. Suitable compounds include compounds which interact with G-protein coupled receptors, adenylate cyclase antagonists, cAMP inhibitors, and cAMP phosphodiesterase agonists. The associated method comprises contacting a cell with one or more such agents, preferably small molecules, in an amount sufficient to reverse or control the aberrant growth state, e.g., to antagonize a normal ptc pathway, agonize a normal hedgehog pathway, or agonize smoothened activity.
In one embodiment, the invention relates to a method for inhibiting an altered growth state of a cell having a ptc loss-of-function phenotype or a smoothened gain-of-function phenotype, by contacting the cell with a ptc agonist in a sufficient amount to inhibit the altered growth state, wherein the ptc agonist is a organic molecule having a molecular weight less than about 750 amu.
In another embodiment, the invention relates to a method for inhibiting aberrant proliferation of a cell having a ptc loss-of-function phenotype or a smoothened gain-of-function phenotype by contacting the cell with a ptc agonist in a sufficient amount to inhibit proliferation of the cell.
In certain embodiments, the ptc agonist causes repression of smoothened-mediated signal transduction.
In certain embodiments, the ptc agonist is a steroidal alkaloid.
In certain embodiments, the steroidal alkaloid is represented in the general formulas (I), or unsaturated forms thereof and/or seco-, nor- or homo-derivatives thereof: 
wherein, as valence and stability permit,
R2, R3, R4, and R5, represent one or more substitutions to the ring to which each is attached, for each occurrence, independently represent hydrogen, halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, xe2x95x90O, xe2x95x90S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or xe2x80x94(CH2)mxe2x80x94R8; 
R6, R7, and Rxe2x80x27, are absent or represent, independently, halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, xe2x95x90O, xe2x95x90S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or xe2x80x94(CH2)mxe2x80x94R8, or
R6 and R7, or R7 and Rxe2x80x27, taken together form a ring or polycyclic ring, e.g., which is substituted or unsubstituted,
with the proviso that at least one of R6, R7, or Rxe2x80x27 is present and includes a primary or secondary amine;
R8 represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle, or a polycycle; and
m is an integer in the range 0 to 8 inclusive.
In particular embodiments,
R2 and R3, for each occurrence, is an xe2x80x94OH, alkyl, xe2x80x94O-alkyl, xe2x80x94C(O)-alkyl, or xe2x80x94C(O)xe2x80x94R8;
R4, for each occurrence, is an absent, or represents xe2x80x94OH, xe2x95x90O, alkyl, xe2x80x94O-alkyl, xe2x80x94C(O)-alkyl, or xe2x80x94C(O)xe2x80x94R8;
R6, R7, and Rxe2x80x27 each independently represent, hydrogen, alkyls, alkenyls, alkynyls, amines, imines, amides, carbonyls, carboxyls, carboxamides, ethers, thioethers, esters, or xe2x80x94(CH2)mxe2x80x94R8, or
R7, and Rxe2x80x27 taken together form a furanopiperidine, such as perhydrofuro[3,2-b]pyridine, a pyranopiperidine, a quinoline, an indole, a pyranopyrrole, a naphthyridine, a thiofuranopiperidine, or a thiopyranopiperidine
with the proviso that at least one of R6, R7, or Rxe2x80x27 is present and includes a primary or secondary amine;
R8 represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle, or a polycycle, and preferably R8 is a piperidine, pyrimidine, morpholine, thiomorpholine, pyridazine,
In certain embodiments, the steroidal alkaloid is represented in the general formula (II), or unsaturated forms thereof and/or seco-, nor- or homo-derivatives thereof: 
wherein R2, R3, R4, R5, R6, R7, and Rxe2x80x27 are as defined above, and X represents O or S, though preferably O.
In certain embodiments, the steroidal alkaloid is represented in the general formula (III), or unsaturated forms thereof and/or seco-, nor- or homo-derivatives thereof: 
wherein
R2, R3, R4, R5 and R8 are as defined above;
A and B represent monocyclic or polycyclic groups;
T represent an alkyl, an aminoalkyl, a carboxyl, an ester, an amide, ether or amine linkage of 1-10 bond lengths;
Txe2x80x2 is absent, or represents an alkyl, an aminoalkyl, a carboxyl, an ester, an amide, ether or amine linkage of 1-3 bond lengths, wherein if T and Txe2x80x2 are present together, than T and Txe2x80x2 taken together with the ring A or B form a covelently closed ring of 5-8 ring atoms;
R9 represent one or more substitutions to the ring A or B, which for each occurrence, independently represent halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, xe2x95x90O, xe2x95x90S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or xe2x80x94(CH2)mxe2x80x94R8; and
n and m are, independently, zero, 1 or 2;
with the proviso that A and R9, or T, Txe2x80x2 B and R9, taken together include at least one primary or secondary amine.
In certain embodiments, the steroidal alkaloid is represented in the general formula (IV), or unsaturated forms thereof and/or seco-, nor- or homo-derivatives thereof: 
wherein
R2, R3, R4, R5, R6 and R9 are as defined above;
R22 is absent or represents an alkyl, an alkoxyl or xe2x80x94OH.
In certain embodiments, the steroidal alkaloid is represented in the general formula (V) or unsaturated forms thereof and/or seco-, nor- or homo-derivatives thereof: 
wherein R2, R3, R4, R6 and R9 are as define above;
In certain embodiments, the steroidal alkaloid is represented in the general formula (VI), or unsaturated forms thereof and/or seco-, nor- or homo-derivatives thereof: 
wherein R2, R3, R4, R5 and R9 are as defined above;
In certain embodiments, the steroidal alkaloid is represented in the general formula (VII) or unsaturated forms thereof and/or seco-, nor- or homo-derivatives thereof: 
wherein R2, R3, R4, R5 and R9 are as defined above.
In certain embodiments, the steroidal alkaloid does not substantially interfere with the biological activity of such steroids as aldosterone, androstane, androstene, androstenedione, androsterone, cholecalciferol, cholestane, cholic acid, corticosterone, cortisol, cortisol acetate, cortisone, cortisone acetate, deoxycorticosterone, digitoxigenin, ergocalciferol, ergosterol, estradiol-17-xcex1, estradiol-17-xcex2, estriol, estrane, estrone, hydrocortisone, lanosterol, lithocholic acid, mestranol, xcex2,-methasone, prednisone, pregnane, pregnenolone, progesterone, spironolactone, testosterone, triamcinolone and their derivatives.
In certain embodiments, the steroidal alkaloid does not specifically bind a nuclear hormone receptor.
In certain embodiments, the steroidal alkaloid does not specifically bind estrogen or testerone receptors.
In certain embodiments, the steroidal alkaloid has no estrogenic activity at therapeutic concentrations.
In certain embodiments, the ptc agonist inhibits ptc loss-of-function or smoothened gain-of-function mediated signal transduction with an ED50 of 1 mM or less.
In certain embodiments, the ptc agonist inhibits ptc loss-of-function or smoothened gain-of-function mediated signal transduction with an ED50 of 1 xcexcM or less.
In certain embodiments, the ptc agonist inhibits ptc loss-of-function or smoothened gain-of-function mediated signal transduction with an ED50 of 1 nM or less.
In certain embodiments, the cell is contacted with the ptc agonist in vitro.
In certain embodiments, the cell is contacted with the ptc agonist in vivo.
In certain embodiments, the ptc agonist is administered as part of a therapeutic or cosmetic application.
In certain embodiments, the therapeutic or cosmetic application is selected from the group consisting of regulation of neural tissues, bone and cartilage formation and repair, regulation of spermatogenesis, regulation of smooth muscle, regulation of lung, liver and other organs arising from the primative gut, regulation of hematopoietic function, regulation of skin and hair growth, etc.
In another aspect, the invention relates to a pharmaceutical preparation comprising a steroidal alkaloid represented in the general formulas (I), or unsaturated forms thereof and/or seco-, nor- or homo-derivatives thereof: 
wherein, as valence and stability permit,
R2, R3, R4, and R5, represent one or more substitutions to the ring to which each is attached, for each occurrence, independently represent hydrogen, halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, xe2x95x90O, xe2x95x90S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or xe2x80x94(CH2)mxe2x80x94R8;
R6, R7, and Rxe2x80x27, are absent or represent, independently, halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, xe2x95x90O, xe2x95x90S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or xe2x80x94(CH2)mxe2x80x94R8, or
R6 and R7, or R7 and Rxe2x80x27, taken together form a ring or polycyclic ring, e.g., which is substituted or unsubstituted,
with the proviso that at least one of R6, R7, or Rxe2x80x27 is present and includes a primary or secondary amine;
R8 represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle, or a polycycle; and
m is an integer in the range 0 to 8 inclusive.
In another aspect, the invention provides a method for inhibiting an altered growth state of a cell having a ptc loss-of-function phenotype, hedgehog gain-of-function phenotype, or a smoothened gain-of-function phenotype, by contacting the cell with a composition including a cAMP agonist.
In certain embodiments, a cAMP agonist activates adenylate cyclase.
In certain embodiments, a cAMP agonist is a cAMP analog.
In certain embodiments, a cAMP agonist is a cAMP phosphodiesterase inhibitor.
In certain embodiments, the composition may include more than one cAMP agonist.
In certain embodiments, the composition inhibits ptc loss-of-function, hedgehog gain-of-function, or smoothened gain-of-function mediated signal transduction with an ED50 of 1 mM or less.
In certain embodiments, the composition inhibits ptc loss-of-function, hedgehog gain-of-function, or smoothened gain-of-function mediated signal transduction with an ED50 of 1 xcexcM or less.
In certain embodiments, the composition inhibits ptc loss-of-function, hedgehog gain-of-function, or smoothened gain-of-function mediated signal transduction with an ED50 of 1 nM or less.
In certain embodiments, the cell is contacted with the composition in vitro.
In certain embodiments, the cell is contacted with the composition in vivo.
In certain embodiments, the composition is administered as part of a therapeutic or cosmetic application.
In certain embodiments, the therapeutic or cosmetic application is selected from the group consisting of regulation of neural tissues, bone and cartilage formation and repair, regulation of spermatogenesis, regulation of smooth muscle, regulation of lung, liver and other organs arising from the primative gut, regulation of hematopoietic function, regulation of skin and hair growth, etc.
In certain embodiments, the composition includes forskolin or a derivative thereof.
In yet another aspect, the invention relates to a method for treating or preventing basal cell carcinoma, comprising administering a composition including a cAMP agonist to a patient in an amount sufficient to inhibit progression of basal cell carcinoma.
In still another aspect, the invention relates to a method for inhibiting an altered growth state of a cell having a ptc loss-of-function phenotype, hedgehog gain-of-function phenotype, or a smoothened gain-of-function phenotype, by determining the phenotype of the cell; and, if the phenotype is a ptc loss-of-function, hedgehog gain-of-function, or a smoothened gain-of-function phenotype, treating the cell with a cAMP agonist in an amount sufficient to inhibit the altered growth state of the cell.